Respiratory tract infections are caused by a variety of microorganisms. Such infections have a myriad of consequences for the community including increased treatment burden and cost, and for the patient in terms of more invasive treatment paradigms and potential for serious illness or even death. It is unfortunate that deliberate infections by lethal inhaled microorganisms have been used in attacks against humans and continue to be a serious security threat. An effective broad-spectrum prophylaxis and treatment against such threats would be very valuable.
As an example of a serious health care burden, high rates of pulmonary colonization with Pseudomonas aeruginosa and the difficulty in eradicating those infections, can lead to declines in lung function, increased number and/or frequency of exacerbations, increased hospitalization and a general decline in health in patients with cystic fibrosis (CF), non-CF bronchiectasis, and patients suffering from severe Chronic Obstructive Pulmonary Disease (COPD). Other lung infections including non-tuberculous mycobacteria (NTM) infections also have the potential to increase morbidity and mortality. These issues have necessitated a search for safe and effective inhaled antibiotics to more effectively treat their lung infections.
There are two currently approved inhaled antibiotics for treating CF infection: the aminoglycoside tobramycin (TOBI), and the monobacatam aztreonam lysine (Cayston). Cayston is approved for thrice-daily inhaled treatment while TOBI is given twice-daily. It would be ideal if there were additional inhaled antibiotic options for CF patients which were more convenient (e.g., once-daily), or more effective, than the currently approved inhaled antibiotics. Additionally, there are no inhaled antibiotics approved for non-CF bronchiectasis patients.
Ciprofloxacin is a fluoroquinolone antibiotic that is indicated for the treatment of lower respiratory tract infections due to P. aeruginosa, which is common in patients with CF and non-CF bronchiectasis. Ciprofloxacin is broad spectrum and, in addition to P. aeruginosa, is active against several other types of gram-negative and gram-positive bacteria. Thus, CF patients who have developed resistance to the aminoglycoside tobramycin (TOBI), or aztreonam lysine (Cayston), can likely still be treated with ciprofloxacin. There is no known cross-resistance between ciprofloxacin and other classes of antimicrobials.
Despite its attractive antimicrobial properties, oral and/or IV ciprofloxacin can produce bothersome side effects, such as GI intolerance (vomiting, diarrhea, abdominal discomfort), as well as dizziness, insomnia, irritability and increased levels of anxiety. There is a clear need for improved treatment regimes that can be used chronically, without resulting in these debilitating side effects. Delivering ciprofloxacin as an inhaled aerosol has the potential to address these concerns by compartmentalizing the delivery and action of the drug in the respiratory tract, which is the primary site of infection.
Currently there is no aerosolized form of ciprofloxacin with regulatory approval for human use, capable of targeting antibiotic delivery direct to the area of primary infection. In part this is because bitterness of the drug has inhibited development of a formulation suitable for inhalation. Furthermore, the drug residence time in the lung is too short to provide additional therapeutic benefit over drug administered by oral or IV routes. Thus, there have been efforts to develop liposomal formulations of ciprofloxacin with improved therapeutic and convenience properties (Yim et al, 2006; Serisier et al, 2013; Bruinenberg et al., 2011; Cipolla et al., 2011, 2013a, 2013b; Cipolla and Chan, 2013).
There are a variety of formulation technologies that have been evaluated for their ability to modulate the release properties of pharmaceutical drugs, or target delivery to specific organs or cells, including liposomes (Drummond et al. 2000). Phospholipid vehicles as drug delivery systems were rediscovered as “liposomes” in 1965 (Bangham et al., 1965). The therapeutic properties of many active pharmaceutical ingredients (APIs) can be improved by their incorporation into liposomal drug delivery systems. The general term liposome covers a wide variety of structures, but generally all are composed of one or more lipid bilayers enclosing an aqueous space in which drugs can be encapsulated.
Liposome encapsulation improves biopharmaceutical characteristics through a number of mechanisms including altered drug PK and biodistribution, sustained drug release from the carrier, enhanced delivery to disease sites, and protection of the active drug species from degradation. A wide variety of drugs have been formulated into liposomes including small molecules, peptides, and nucleic acids; hydrophilic drugs are generally dissolved in the aqueous compartment while hydrophobic drugs are associated with the lipid bilayers (Drummond et al. 2000, Cipolla et al. 2013b). Liposome formulations of the anticancer agents doxorubicin (Myocet®/Evacet®, Doxyl®/Caelyx®), daunorubicin (DaunoXome®), and vincristine sulfate (Marquibo®), the anti-fungal agent amphotericin B (Abelcet®, AmBisome®, Amphotec®) and a benzoporphyrin (Visudyne®) are examples of successful products introduced into the US, European and Japanese markets over the last two decades. The proven safety and efficacy of lipid-based carriers make them attractive candidates for the formulation of pharmaceuticals.
The physicochemical properties of liposomes, and in particular their drug release profile, can be engineered into the formulation via a variety of strategies including: the liposomal composition (e.g., an increase in the acyl chain length of phosphatidylcholine (PC) reduced the release rate of liposomal vincristine (Boman et al. 1993)), the presence and concentration of sterol (e.g., the addition of 30% cholesterol reduces membrane permeability leading to a slower drug release rate for many liposomal formulations), surface modification with polyethyleneglycol (PEG) (e.g., doxorubicin liposomes containing PEG had longer circulation half-lives and slower release than egg PC/cholesterol liposomes (Abraham et al. 2005)), liposomal size and lamellarity (e.g., unilamellar liposomes typically release their contents at a faster rate than multilamellar vesicles), the drug to lipid ratio (e.g., higher drug to lipid ratios reduced the release rate of liposomal vincristine, and were also found to increase its efficacy (Johnston et al. 2006)), the state of the drug inside the vesicle (e.g., liposomes containing precipitated doxorubicin had slower release than those with doxorubicin in solution (Lasic et al. 1995)), the choice of drug loading method (e.g., a larger transmembrane pH gradient reduced the release rate of liposomal doxorubicin (Mayer et al. 1990)), and other factors including osmolarity, pH, and choice of buffer and excipients. We were interested to see if the encapsulation state and release properties of a liposomal ciprofloxacin formulation could be modified by addition of surfactants.
A number of US patents describe liposomal formulations for inhalation: U.S. Pat. Nos. 8,414,915; 8,268,347; 8,119,156; and 8,071,127. These patents describe formulations of liposomal ciprofloxacin and mixtures of liposomal ciprofloxacin with free ciprofloxacin. These liposome formulations were designed to be robust to the nebulization process such that the encapsulation of drug was principally unaltered. In comparison to the current oral or IV ciprofloxacin formulations, a liposomal ciprofloxacin aerosol formulation should offer several benefits: 1) higher drug concentrations in the lung, 2) increased drug residence time via sustained release at the site of infection, 3) decreased side effects, 4) increased palatability, 5) better penetration into the bacteria, and 6) better penetration into the cells infected by bacteria. It has previously been shown that inhalation of liposome encapsulated fluoroquinolone antibiotics may be effective in treatment of lung infections and in a mouse model of F. tularensis liposomal encapsulated fluoroquinolone antibiotics were shown to be superior to the free or unencapsulated fluoroquinolone by increasing survival (CA2,215,716, CA2,174,803, and CA2,101,241).
Another group of US patents describe inhaled formulations of liposomal aminoglycosides for treatment of lung infections: U.S. Pat. Nos. 8,226,975, 7,879,351, and 7,718,189.
However, there remain opportunities to develop novel liposomal formulations with improved properties and release characteristics, one of those being the ability to modify their properties in a simple and flexible manner. We describe a strategy that we have used to develop these new liposomal formulations through the addition of surfactant. The addition of surfactant to the pre-existing liposomal formulation, when properly designed and executed, can allow for the amount of free drug and the release profile to be tailored into the product.
There is an extensive history of detergents being used to solubilize biological membranes to allow for elucidation of membrane structure and function (Helenius and Simons 1975). The ability of surfactants to solubilize phospholipids, specifically, was reviewed (Lichtenberg et al. 1983). Typically, as surfactant is added to phospholipids, surfactant initially partitions between the solution and the phospholipid bilayers and the bilayer permeability may increase without loss of structure (Lichtenberg et al. 1983). Once the phospholipid bilayers become saturated with surfactant, addition of more surfactant leads to the formation of mixed micelles of surfactant and phospholipid until all of the remaining surfactant-saturated bilayers are converted to mixed micelles. Any further addition of surfactant leads to a decrease in the size of the micelles as they become more dilute in phospholipid content. Vesicle-surfactant systems have frequently been characterized by monitoring their light scattering properties, with maximum turbidity generally associated with the surfactant saturated bilayer state (Velluto, Gasbarri et al. 2011) followed by a rapid decline in turbidity once the bilayers are completely solubilized by surfactant (Paternostre et al. 1988, Ribosa et al. 1992, Lasch 1995, Cho et al. 1999, Deo and Somasundaran 2003).
Liposomes have been used as a simplified model of biological membranes. Interest in the development of antimicrobials that would function by disruption of the bacterial membrane spurred a better understanding of the interaction of surfactant-like molecules with liposomes (Nagawa and Regen 1992, Liu and Regen 1993). In addition, liposomes were being investigated as drug delivery vehicles so knowledge of the factors which affected the timing and rate of release of the encapsulated drug is paramount. After in vivo administration, liposomes come into contact with many natural amphiphiles present in physiological fluids. Surfactants can be used as a simplification to the complex biological milieu, allowing for characterization of drug release from liposomes in the presence of surfactant (Ruiz et al. 1988).
Two release mechanisms were identified for liposome-encapsulated carboxyfluorescein (CF) in response to added surfactant (Nagawa and Regen 1992). For some combinations of liposomes and surfactant, there was a gradual release of the encapsulated agent from all vesicles with increasing surfactant concentration. For other combinations, there was a catastrophic rupture in which a subset of the vesicles rapidly released their entire encapsulated payload while others were unaffected. These findings were expanded by showing that Triton X-100 in its monomeric form (below its CMC) induced leakage of CF from all liposomes that were studied, but when Triton X-100 was in its micellar form (above its CMC) it was able to rupture only gel-phase vesicles (below the Tm) and compact fluid-phase vesicles (i.e., those containing significant cholesterol) but not fluid-phase liposomes (Liu and Regen 1993). The properties of the encapsulated agent can also influence its release behavior. More surfactant was required to release an equivalent percentage of a larger molecule, dextran, versus a smaller molecule, glucose (Ruiz, et al. 1988). Finally, the kinetics of release of CF using sublytic concentrations of Triton X-100 was explored for both sonicated and extruded liposomes composed of egg or soy PC (Memoli et al. 1999). In both cases, release of CF was almost instantaneous and reached a stable value within a few minutes, suggesting that transient holes were formed upon association of low levels of surfactant with liposomes but these holes then closed limiting further release of CF (Memoli et al. 1999). In contrast, upon addition of solubilizing levels of surfactant, there was a complete breakdown of liposome structure within 0.2 sec with complete release of encapsulated drug (Velluto et al. 2011). While interactions of surfactants with liposomes have been more fully explored from a mechanistic basis, there has been little interest in utilizing this property of surfactants to modify the encapsulation state and release properties of liposomes for therapeutic purposes.
We describe the development of new liposomal formulations of ciprofloxacin containing surfactants to modify their release properties. Liposomal ciprofloxacin is in development as a once-daily inhaled antibiotic to treat respiratory infections in indications such as cystic fibrosis (CF) and non-CF bronchiectasis (Bruinenberg et al., 2011, Cipolla et al., 2011, Cipolla and Chan, 2013, Cipolla et al., 2013b, Serisier et al., 2013, Yim et al., 2006); it also appears to be effective against a variety of potential bioterrorism infections including tularemia (Conley et al. 1997) and plague (Hamblin et al., 2013). In contrast, currently approved inhaled antibiotics for cystic fibrosis are not encapsulated in liposomes and must be administered twice- or thrice-daily (Cipolla and Chan, 2013). These antibiotics failed to show adequate efficacy and safety for approval outside cystic fibrosis (Cipolla and Chan, 2013). There are two liposomal ciprofloxacin formulations being investigated in the clinic: Lipoquin™, for which all of the ciprofloxacin is encapsulated into liposomes and a combination formulation, termed Pulmaquin™ which contains both a mixture of free, unencapsulated ciprofloxacin and liposome-encapsulated ciprofloxacin. We were motivated to determine if we could apply the inventive step to modify the Lipoquin formulation specifically through the addition of surfactant to create a mixture of both free and encapsulated ciprofloxacin in varying portions, and which may possess modified release properties compared to Lipoquin or Pulmaquin. Additionally, we investigated whether such formulations could be designed to retain their physical properties after long term refrigerated storage as well as after nebulization to create an inhalation aerosol. We also believe that the learnings from the addition of surfactant to liposomal ciprofloxacin formulations can be broadly applied to liposomal systems in general and to treat a wide variety of indications, not limited to lung infections or administration through the inhalation route.